Our laboratory investigates mechanisms of cell fate specification along the animal?vegetal (A?V) axis of the sea urchin (Strongylocentrotus purpuratus) embryo. Our major focus is to understand the gene regulatory networks (GRNs) and signaling pathways that specify ectodermal domains, beginning with pre-ectoderm and the subsequent aboral, oral, neural and ciliagenic territories. SoxB1 and nuclear beta-catenin cross-regulatory mechanisms. Previously we showed that the SoxB1transcription factor is a key regulator of mesoderm and endoderm because it antagonizes function of nuclear beta-catenin, which is the initial activator of the endomesoderm (EM) GRN. Thus, tight regulation of the Sox B1/beta-catenin ratio is critical for allocating different cell fates to early blastomeres. Regulation of SoxB1 levels is achieved by transcriptional repression and, surprisingly, spatially regulated turnover of SoxB1 protein (Angerer et al., 2005; experimental work was completed before arriving at NIDCR and the publication was finished shortly after). The aims of this subproject are to understand the molecular mechanisms by which SoxB1 antagonizes nuclear beta-catenin and, conversely, the pathways that lead from nuclear beta-catenin to SoxB1 transcriptional repression and turnover. Mechanisms of specification of primitive neurons at the embryo?s animal pole. A second subproject seeks to define the GRN underlying specification of cells at the animal pole of the sea urchin embryo [Animal Pole Domain (APD)], which will form part of the larval nervous system and ciliated band. This region of presumptive ectoderm is unique in that it is the most resistant to alteration of cell fates by mis-expression of genes that promote EM differentiation. In contrast, in the absence of beta-catenin-dependent EM signals, almost the entire ectoderm differentiates with an APD-like fate. Thus, APD-specifying genes are repressed by EM signals in all ectodermal cells, except those at the animal pole. This, and other evidence suggest that some APD cells may initially be specified by maternal molecules. We plan to define the core GRN of these primitive neurons, especially maternal and zygotic factors that initiate neuronal specification. Two different approaches have been used to attack this problem. The first, under extramural funding (GM2553-25) at the University of Rochester, was a cDNA subtractive hybridization screen to select mRNAs up regulated when the EM GRN was completely inactivated. Selected sequences are depleted of those involved in housekeeping functions and differentiation of most tissues and enriched in mRNAs involved in APD specification and differentiation. Selected and normal embryo cDNAs were used to identify, in macroarrays of ~ 100,000 cDNA clones, those that represent mRNAs strongly enriched in the selected cDNA population. Analysis of these continued this year at the NIH. From the initial screen, the most interesting genes expressed in the APD are: 1) FoxQ2, a novel winged helix transcription factor, 2) at least one new tolloid/BMP1-like astacin protease, 3) a matrix metalloprotease, most closely related to a neural metalloprotease, 4) a neural-specific molecular chaperone, PACRG. Unexpectedly, we identified some mRNAs expressed exclusively at both poles of the embryo. The protease genes are expressed in a few cells scattered throughout the APD at very early stages, suggesting that they identify neural precursors. The second approach initiated this year at NIDCR, was to survey the expression of genes whose homologs in other systems have neurogenic gene regulatory functions. We used the newly completed S. purpuratus genome sequence and computational tools (see below) to rapidly identify homologs of genes encoding neurogenic transcription factors. Of 16 such genes, 11 are expressed in early development. Most interesting are 10 expressed in the absence of intercellular signals and 7 expressed in the absence of nuclear beta-catenin function, suggesting that they are activated cell-autonomously by maternal factors. mRNAs for at least 6 of these accumulate in the APD and 4 (FoxQ2, retinal anterior homeobox, achaete-scute and SoxC) appear very early, consistent with their functioning at the top of the neurogenic GRN. Their functions will be established by morpholino antisense knockdowns. TGF-beta signaling in endoderm development. In a third sub-project, Aditya Sethi showed that specific inhibition of the Alk4 receptor, which transduces a subset of TGF-beta signals, blocks normal development of early ectoderm and endoderm. In addition to confirming that signaling from the TGF-beta family member, nodal, specifies oral ectoderm, he found that an additional signal(s) is required for timely endoderm specification and correct gastrulation movements. By analyzing endodermal and ectodermal marker gene expression using in situ hybridization, immunohistochemistry and real time PCR assays, he found that the new signal may correspond to the long-sought, but still undefined ?early signal? required for EM development. After mining the genome with the new computational tools developed in our lab (see below), he identified 6 TGF-beta candidates. The expression of only one of these, activin B, is appropriate for its being the identified signal. He will test whether activin B promotes endoderm development by eliminating it with a morpholino. If it does, he will examine whether it signals through Alk4 by testing whether endoderm development can be rescued with constitutively active Alk4 receptor. Development of resources and tools for mining and annotation of the sea urchin genome. This year Zheng Wei developed computational tools for analyzing the sea urchin genome. He converted whole S. purpuratus genome sequence into a database of predicted peptides and then constructed a relational database that linked individual peptides (approximately 40,000) to gene names, expressed sequence tags and cDNA sequences available in the public databases. This effort produced a gene list, one of three currently being used to annotate the sea urchin genome. Dr. Wei also created, and made publicly available on our web site, a set of very user-friendly computational tools for identifying genes, which are being used by the international consortium of laboratories working on the annotation project. We will continue our annotation efforts by creating a whole-genome microarray with information obtained from our gene list. Dr. Wei has identified oligonucleotides representing more than 20,000 individual genes for this microarray, the first in this system. We will use this microarray initially for temporal expression profiling and then create a second smaller microarray of developmentally regulated embryonic genes, which will be made publicly available for experimental analyses of development.